Gene Targeting by Homologous Recombination in Embryonic Stem Cells

Abstract

This chapter discusses gene targeting by homologous recombination in embryonic stem (ES) cells. ES cells are undifferentiated totipotent cells. They derive from the inner cell mass of a mouse blastocyst and can be perpetuated in culture. ES cells should be cultured on a feeder layer of inactivated embryonic fibroblasts. It is important that the ES cells be trypsinized to obtain a single cell suspension to avoid differentiation. The targeting construct consists of a genomic fragment, from which exonic regions coding for essential parts of the gene have been removed and substituted by a neomycin cassette. The targeting frequency is variable depending on each particular locus. For some loci, the basic targeting strategy described in the chapter gives an acceptable targeting frequency. However, for some loci the frequency may be too low to identify the homologous recombinants without enriching strategies.